CA1199290A - Culture and process for producing a41030 antibiotics - Google Patents

Abstract

Abstract of the Invention A hitherto undescribed microorganism, Streptomyces virqiniae NRRL 12525, which upon culturing produces the A41030 antibiotic complex comprising several factors. The A41030 antibiotics produced show anti-bacterial activity against the gram-positive genera Staphylococcus and Streptococcus which are resistant to penicillin. In addi-tion, these antibiotics act to promote growth and improve feed efficiency in ruminant animals, poultry and swine, and other livestock, and to improve milk production in ruminant animals.

Description

CULTURE AND PROCES~ FOR PRODUCING A~1030 ANTIBIOTICS

Summar~ of the Invention This invention relates to an axenic culture of the microorganism Streptomyces virginiae NRRL 12525, and to the use of NR~L 12525 for the production of the anti-biotic A41030 complex, which is comprised of individual factors A, B~ C, D, E, F, and G. This complex is produced by culturing the hitherto undescribed microorganism~
Streptomyces virginiae NRRL 12525, or an A41030-producing mutant or variant thereof r under submerged aerobic fermen-tation conditions9 These A41030 antibiotics inhibit the growth of certain pathogenic microorganisms, in particular, those within the gram-positive genera Staphylococcus and Streptococcus which are resistant to penicillin. The antibiotics of this invent~on act to promote growth and improve feed efficiency in ruminant animals, poultry and swine, and other livestock~ and to improve milk production in ruminant anim~lsO

Description of the Drawings ~0 Infrared absorption ~pectra of A41030 factors A, B, C, D, E, F, and G are presented in the drawings as follows:
Figure 1 - A41030 factor A (in KBr pellet) Figure 2 ~ A41030 factor B (in KBr pellet) Figure 3 - A41030 factor C ~in KBr pellet) Figure 4 - A41030 factor D (in KBr pellet) Figure 5 - A41030 factor E (in KBr pellet) Figure 6 - A41030 factor F (in KBr pellet) Figure 7 - A41030 factor G (in KBr pellet) ~ ~ ~3~

Detailed Description of the Invention This invention relates to an axenic culture of the hitherto undescribed microorganism, S~reptomyces virginiae NRRL 12525, which produces an antibiotic complex comprising several factors, including individual factors A, B, C, D, E, F, and G~ For convenience, this culture has been desig-nated in our laboratory as culture A41030.4.
The term "complex", as used in the Eermentation artr and in this specification, refers to a mixture of coproduced individual antibiotic factors. As will be recognized by those familiar with antibiotic production by fermentation, the number and ratio of the individual factors produced in an antibiotic complex will vary, depending upon the fermentation conditions and the strain used.
Culture A41030.49 which is a chemically-induced mutant of a strain of the Streptomyces virginiae culture which was initially isslated from a soil sample collected in Indianapolis, Indiana, has been deposited and made a part of the stock culture collect.ion of the Northern Regional Research Cent~r, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public unde.r the number NR~J 125250 ~i ~ " J

;d ~ ~

Culture A41030.4 was ob~ained by treatment of culture A41030 with mitomycin C and N-methyl--N~-nitro-N-nitrosoguanidine~
It will be recognized by those skilled in the art that it would be possible to generate additional strains which have essentially the same biosynthetic capabilities as Streptomyces virginiae NRRL 12525, by subjecting culture A41030 to other mutagenic treatments.
In addition to mitomycin C, other suitable agents include acriflavines, acridine orange, ethidium bromide, and similar chemical agents. Although N-methyl-N' nitro-N
nitrosoguanidine was used, along with mitomycin C, to obtain NRRL 12525, other known mutagens such as ultra~
violet rays, X-rays, high-frequency rays, radio-active rays and other chemical agents could be used to induce a similar mutagenesis.
The classification of Culture A41030.4 as a chemically-induced mutant of a strain of Streptomyces virginiae, i5 based upon a simultaneous culturing of Streptomyces avidinii ATCC 27419; Streptomyces columbiensis ATCC 27425; Streptomyces goshikiensis ATCC 23914; Streptomyces griseolavendus ATCC 25457;
Streptomyces lavendulae ATCC 8664; Streptomyces toxytricini ATCC 19813; and Streptomyces virginiae, Gr undy, Whi tman, Rdzok, Hanes and Sylvester 1952, ATCC 19817. The methods and media recommended by Shirling and Gottlieb ["Methods of Characterization of StreptQmyces species," Int~ J. SystO Bacteriol. 16(3), 313-340 (1966)], along with certain supplementary tests were used. Culture A41030.4 was also compared with published descriptions of the above-named strains appearillg in "Bergey~s ~. ~
-'"!

x-ssa~ ~4~

Manual of Determinative Bacteriology" (8th Edition, edited b~ R~E. ~uchanan and N.,E. Gibbons, The Willia~s and Wilkins Co., Baltimore! k1aryland~; and by 5hlrli~g and Got~lieb, "Cooperative Description of Type Stxains of Stxeptomyces"~ Int. J. Syst. Bacteriol. 18(2), 17 (l968)-5i~ce Culture A41030~4 produces no aerialmycelia and no spores on any ~edium, it di~fers from all the above-named species.
1~ CHARACTERIZATION OF A41030.4 CULTVRE
Morphologv Produces no a~riaI mycelia and no spores~
Cultural Chaxacterlstics The growth characteris.ics of culture ~41030.4 on ~arious media are presented in the ollow-lng Table 1, Color names were assigned according to the ISCC-N~S Centroid Color Chart~ Standard SampLe ~o. 2106 (~.~ational ~ureau of Standards, U.S. Department G ~ Com-merce, 1958), and the Color ~armony i~nual, 4th Edition (Color Standards Department, Contain~?r Corpcratior. of America, Chicago, Illinois, lgS3).

~5 ~Q

.~

~1~3~
X-5984 ~5-Cultural Charact:eristics on Various Medi~
Medi~n Charaeteris~ics Yeast extract malt extract Good growth, rever~e ~O, agar ~ISP medium ~2) gy.Y; no aerial myceliu~;
~o solu'ole pigmen~
~atmeal agar (I5~ medium Poor growth, reverse 93~
1~ ~3) vGra~; no aerial mycelium, no soluble pigment.
Inorganio s~lts-sta~ch Poox growtn, revers~ 89.
asar ~ISP medium #4) p. Y; ilO aerial mycelium;
~ no soluble pigment.
Glycerol aspar~gine asar ~ Poor growth, revere 89.
~ISP medi~m ~5) p. Y; no aerial mycelium;
no solu~le pigment.
Tomato pa.ste Good growth, rever3e 54.
oa~meal agar brO; no aerial ~ycelium;
no soluble pigmer.t.
Th~ Cu'ture A41030r4 was studied for selected physiological properti s in accord~nce wlth sta~dard pxocedures. The prcperties obser~ed and characteristics ~-

2~ found are re~oxded in Table ~, which rollows:

3~ .

.:.......... ,...................... .,,.,.. ,........... .. ,~,.

w ~ ~ ~ ~ x o ~BLF. ~
PHYSIOLOGIC'AL PROP~RTIES OF A41030.4noid-like P~m~nt Procluction on:
l~ Tryptone yeast extractMelanoid~ e pi~ment : ~roth ~I~P #1~
. 7~ Peptone yeast eY.tractMeLanoid-like pigment - iron agar slants ~ISP ~6) 3. Tyrosine agar slants NG rnelanoid-like pigment . ~SP #73 Nitrate Re~uc~ioll Negative reaction Cela~in Liquefac~ion Negative reactlon NaCi Tol~rance S~a~-ch ~yd~olysis Negative reactiQn Skim Milk ~ Partial hydrolysis -- Temperature ~equirements 10 34C.

X~5984 ~7~

A comparison of the carbon utilization ~at-terns of ~ulture A41030.4 and Streptomyces vir~iniae ATCC 19817 was conducted using ISP No. ~ basal medium to which filtex~s~erilized carbon sources were added to equal a final concentration of 1.~%. Plates were read after fourteen days incu~ation at 30C. The results are ~et forth in Table 3, which follows:

~ .

X-5984 -8~

CP~BON UTILIZATICN PATTERNS OF A41030. 4 AND STREPTOMYCl~:S VIR~INIA:E ATCC 198L7 Carbon Source ~41030 . 4 ATCC 19817 Acetate-Na - -D~Ar a~ ino ~ e - -L-Arabinose - -Cellobiose + +
D-Fructose -D C;alactose +
D-Glucose + +
ositol - -LactQse D-~5altose ~ + - s D-l~annitol - -Meli~iose - -Raf f inose - -Rhamno s e ~ -D-Ribose Salicin Succinat~-.Na ~ ~
Sucrose - -D-X~lose - -;2~ Key: - ~ no utilizati3n.
- utilization paxtia 1 u t i 1 i z ~tion ._ 3~

.... . ...... . . . .... ......... .... ... ... . .... . 1 . . . ..... .. . . . .... . . .. ...... . .

X-~U4 ~9~

Uslng hydrclyzed whole ce~lls of the organism, the isomers of diaminopimelic ~cid were de~enmined according to the method of Becker et al., Ap~l. Mic~o-biolO 11, 421-423 ~196~). The results o this study are set fvrth be.low~
T _ Result O~ser~ed Isomers of 2,6- LL-isomer diamino~imel.ic acid A comparison of the similarities and differ-~ ences betwe~n Cultuxe A41030.~ and Strepto~yc svirsiniae ATCC 19817 i~ set forth in Table ~ r ~hich ollows:

1~ COMPA~ISON OF CU~TURE A41030.4 AND S. VIRGINI~E ATCC 19817 Similaxities Differenc~s Carbon utilization pattern Aerial spore mass 20 Melanoid pigment on ISP #6 Fruotose ukilization No melanoid pigment on ISP #7 Galactose utilizatior.
No solublè pigment produc~ion G~latin li~uefaction ~itrate ~Pduction negative ~elanoid ~igments in ISP 1,~1 Rev~rse color ~1orphology S~im milk reactlon NaCl tolerance Starch reduc~ion negati~e Ribo~e utilization Temperature range 3n , . . . ... ...... ............................. . ... ....................... . ... ........... ............................. .
.. ,...... .......... ~.. . ... ....... ............. .................. .. ...................... ............. .. ........................

?~

~-5~84 10-The antibiotic substances of th.is invention are arbitrarily desi~nated herein as A41030 antibi-otics. The A41030 complex contains several individual factors ~hich are designated A41030 fac'ors A, B, Cl D, E, F, an~ Go In discussions of utili y, the term "A41030 ar.tibiotic" will be used, for the sake of brevity, to denote a member selected from the group consisting o~ A41030 comple~, and A41030 ~actors A, B, C~ Dr ~, F, and G.
~s many as seven antibiotic factors ar2 re-1~ covered from the fermentation and are obtained ~s amixkure, the A41030 complex. It will be recognized that the ratio of the facto.s in the A41030 complex will vary, depending upon the ferm~Qntation condltions us~d. The individual factors A, B, C, D, E, Fr and ~
are .~epara~ed and i~olated as individual ~ompo~nds, as hereinafter described. The A41030 complex is solu~le in water, dilute aqueous acid, dilute a~ueous has~, methanol-watPr mixtures, ethanol-water mixtures, dimethylformamide and dimethylformamide-water mlxtures, 2~ dime~hylsulfoxide, dimethylsulfoxide-water mix.ures, ace~onitrile, acetone-, ethyl acetate, tetrahydro uran, methylene chloride, and the like.
The following paragraphs describe the physi-cal and spectral properties o the ~1030 ~'actors which ~5 have thus ~ar been cha~acterized.
A~103a FA~TOR A
Anti~iotic A41030 factor A i5 a whit~, crystalllne solid. Elemental analysis o ~41030 ~actor A indicates .nat it has the following ap~rox~
i.~atQ perc~nA~ase composition: 5~4~.% carbon, 3~53 ... ...... ...... ....... . . . .. .. ..... ................... ............................ .. . ......... . ... ...

hydrog~n, B.ll~ nitrogen, 23.20~ oxysen~ and 8.29~
chlorine. As determined by ield desorption and plasma desorption mass spec-~rometry, A41030 actor A has a molecul~r weisht of 1231. Based on the elemental analysis and the molecular weight, an empirical formula of C58H44C13N7Ol~ is ~ssigned to factor A. Electrometxic titration of factor A in 66~ dimethylformamide in water indicated the presence of three titra~able ~roups having P~a values cf about 5.53, 7.60 and 10.37, with possibly additional pKa's > 10.5 (initial pH 7.83).
Antibiotic A41030 factor ~ has the followins speci~ic rotation: ~a]25 ~19.6 (c, 9.0 in dimethyl~ulfoxide).
The infrared absorption spectr~m of A41030 f~ctor A in K3r pellet is shown in the accomp~nying drawings as ~ig. 1. The follo~ing distinguishable a~sorption maxim~ are observed: 3448-3226 (strong, broad), 1653 Ist_ong!, 1610 (weak), 1587 (m~diumj, 1515 (stron~), 1488 (wea~)~ 1429 (medium)~ 1227 (strons), 1139 (medium), 1064 (strong), and 1010 (strong) cm 1.
The ultraviolet absorption m2xima of A41030 factor A in methanol:water (1:1) under acid, neu~ral, and basic conditions are recorded in Ta~le 5.

~ ' .. ... . . .. ...... .. ~ .... ........ ........ ............... ...... ~ .. ........ . .. ...... .. . . .

~q~

X-5984 -12~

T~BLE 5 W Spectrophotometry c~f A410~0 FactQrs ~actor ~cidic or Neutral B2sic max nm (~) max nm (~) A 278 ~11,100). 298 ~17,200~
278 (9,~0~ ~g~ ,8003 C 278 (8,400) 2g~ ~14,000~
D 278 (10,~00) ~98 (19,900) 1~ E 278 (8,500~ 298 (lS,500) .~ 278 (9,300~ 298 (14,500~
G ~78 (15,000) 298 (18,0003 Antibiotic A41030 factor A is soluble in alcoholOwater mixtures, in dlmethylsulfoY~ide, in ~imethylfo~mamide, in dimethvlsulfoxide-water mixtures, in dimethylformamide-water mixtures, in dilute aqueous acid, and in diiute aaueous base~
on the basis of the observed phys- câl chemical data, the follo~ing structure has bee'n assigned to A4103~ factor A.

~5 .. ............ .. ... ... ..... . .. .. . . .... ............ .. ................ ..... ... ................. . .. . .. .

v q~ 3`~

QH
/ \ /Q\ / ~ / \~/~ \t 110 C ~/IH~N ~C~
/~\ t~ \h c I HO/ ~/
HC~ \()H ~

~ sins a biological assay and high p~rformance liquid chrornatography analysis, it has been round that factor ~ accounts for f rom about 94 to a~out 9 6~ by wei~ht of the antibiotic factors produced by culture A~1030.4, ~ith factors 3, C, D, E, F, and G accountin~
for the remainins a~out 4 to about 6~ by ~eight of the factors produced.
~41030 FAC~OR B . ~.
~ntibiotic A4103Q factor B is a ~hite solid, havins an approximate elemental analysi~ as follows:
5~S4%. carbon, ~o21~ hydro~en, 8.63% nitrog2n, 5.q6 chlo.rine, and by dix~erence, 2~.663 oxygen. E~ectro-metric titration o~ ~actor B in ~6~ dimethylorm~mide 30 in watex indicated the presence of two titratable - ~.. .. .... .. ,.... ~.. , .. ... .. .. ,,.. ,.. , . ,.. - .... - .. .. . - .. -~'53~

groups at p~ values of about 5.6 and 7.5, respectively, with p~ssibly additional pKa's ~10 (initial pH 6.22).
An observed molecular weight of abou~ 1197 was obtained using fast atom bombardment mass spectrometry. Based on elemental analysis and the observed molecular weight~ an empirical formula of C58H45C12N7O18 to factor B.
The infrared absorption spectrum of antibiotic A41030 factor B i~ KBr pellet is shown in the accompanying drawings as FigO 2~ The following distinguishable absorp-tion maxima are observed: 3448-3226 Istrong, broad), 1653 (strong), 1610 (medi~n), 1587 (weak), 1515 (strong), 1488 (weak~, 1429 (medium), 1290 (weak), 1227 (strong), 1139 ~medium), 1064 (strong), and 1010 (strong) cm The ultraviolet absorption maxima of A41030 factor B in neutral, acidic, and basic methanol:water (1:1) are recorded in the foregoing Table 5.
Antibiotic A41030 factor B is soluble in the same solvents as factor A.
On the basis of the ohserved physical chemical data, the following structure has been assigned to A41030 factor B.

~b d X~59~ -15-Q~l I' 'f \I~7/ \7/ 'fi 1 / I\NH~

T/~fi H~ / \OH ~H
~5 A4 10 3 0 E` ACTOR C
Antibiotic A41û30 factor C is a white solid ;~0 ha~ing an approximate elernental analysis as follows:
48 . 87% carbon, 4 . 39% hydrogen, 6 . 169~ nitrogen, 6 0 ~6~
chlorine,- and 33.S1~6 oxygen. ~lectrometric, titration of ~actor C in 66% dimethyllormarnic~e in water indicated ~ha presence or tw~ tltrata~31e groups at P~a values of ;~5 about 5 . 5 arld 7 .1, respectively, with possi}: ly additional PKa ' s ?lQ ~initial pH 6 . 6) . ~n observed mole~ular weight of abcut 1393 was obt~ined usins fast atom bombardment mlass spectrometry. Based on èlernental a~alysis ~nd ' he observed molecular wei~ht, an empirical 3~ 6~H5~Cl3N7~;~3 is assigned to ~actor C

... ........... , .... ...................... ................................... ................................................................... ~.... .

4~

~-59~4 ~1~

The infxared absorption spectrum of anti-biotic A41030 factor C in KBr pellet is shown in the accompanying drawings as Fig. 3. The followiny dis-tinguishable absorption maxima are observed: 3448-322~ (strong, ~road), 1653 (strong), 161Q (medium), 1587 (weak), 15Q4 (strong), 1481 (weak), 1~29 (~edium), 1220 (strong), 1136 (strong), 1064 (weak), 1053 (medi-um), and 1005 (strong) cm 1, The ultraviolet absorption maxima of A41n30 factor C in neutral, acidic, and basic methanol water (1:1) are recorded in Table 5 above.
Antibiotic A41030 factor C is soluble in the same solvents as factor ~.
On the basis of the observed physical chem-ical data, the structur~ of A41030 factor C ls believed 1~ to be as follows:
OH

2~ f ~ C I ~, c I ~ , Crl~
\ O ~/ ~ ''~/ \f=O -HOI~ - fH T I~ \ H\NH2 ;~3 H~ OH ~ ,7a l acl ose) Antibiotic A41030 factor D is a white, amorphous solid having an approximate elemental analysis as follows:
54.46% carbon, 4~35% hydrogen, 7.58~ nitrogen, 4.27%
chlorine, and by difference, 29.34% oxygen. Electrometric titration of factor D in 66% dimethyl-formamide in water indicated the presence of two titratable groups at PKa values of about 5.5 and 7~6, respectively, with possibly additional pKa's >10 (initial pH 6.83)~ An observed molecular weight of about 1326 was obtained using fast a~om bombardment mass spectrometry.
The infrared absorption spectrum of antibiotic A41030 factor D in KBr pellet is shown in the accompanying drawings as Fig. 4. The following distinguishable absorp-tion maxima are observed: 3448-3226 (strong, broad)l 2959 (weak), 1661 (strong), 1592 ~strong), 1511 (strong~, 1429 (weak), 1290 (weak), 1227 (weak), 1212 (medium), 1163 ~weak), 1143 (weak3, 1053 (medium), and 1010 (strong) The ultraviolet absorption maxima of A41030 factor D in neutral, acidic, and basic methanol.water (1:1) are recorded in Table 5 above.
Antibiotic A41030 factor D is soluble in the same solvents as factor A~
On the basis of the observed physical chemical data, the structure of A41030 factor D is believed to be as follows:

~-~984 0~1 C

10 Ho~ ~t ~IH:~

o ~ 1 H
HC~ ~/ QH ~H

plus one or more ~-~utyl groups.
A41030 F~CTO~ E
2~ Antibiotic A41030 factor E i5 a white sQlid having an approximate elemental analysis as follows:
56O06% carbon, 4.06~ hydrogen, 8.53% nitro~en, 3O50~
chlorine, and by di~ference, 27.85~ oxygen~ Electr~-metr.ic titration of factor E in ~6~ dimethylrormamide --in wa~er indicated the presence of two titratable groups at pR~ values of about 5.8 and 7.7 r respec~
~ively, wit~ possibly additional pKa's ~10 (initial pH
6.57~. An ob~erv~d molecular weight of about 1163 w~s obtair.e~ usin~ ~ast atom bombardment mass s2eC~rometry.
3~ A tan~ative em~i-ical formula o~ C58H46ClN7O18 is a~signed to ~actor E.

,.. ,.. :.:
......... ........ ................................... ................................ ................................. ~ .. .......... ....... ........... ...............
. .................. . ..

~a~

~-59~ 19-The infrared absorption spectrum of anti-~iotic A41030 factor E in KBr pellet is shown i~ the accompanying drawings as Fis. 5. The following dis~
tinguishable absorption maxima are observed: 344B-32~6 (strong, b~oad), 1653 (strong), 1600 (medi~m), 15~4 (s~rong), 3429 (w~ak), 1290 (weak)/ 1~38 (medi~m), 113~ (weak), 1064 (weak), and 1010 (strons) cm 1~
The ultraviolet absorption maxima of A41030 factor E in ne~tral, acidic, and basic methanol:water (1 7) are recorded ln Table 5 a~ove.
1~ ~n~i~iotic A41030 ~actor E is soluble in the same solvents as factor A.
Qn the basis of the observed physical chem-ic~l data, the following structure has ~een assigned to A~1030 facto~ E. TH

\~ \ \jj/ \T "a hO ~ h ~ Ch' ~
R~,J~
,I~,D

X 5984 -~0-Antibiotic A41030 factor F is a white ~olid having an approximate elemental analysis ~5 follows~
51.39% carbcn, 3.96% hydrogen, 6.45~ chlorine, 6.45~
nitrogen, an~ 28.65% oxygen. El~ctrometric titration o~ factor F in 66~ dimethtflformamide in wat~r indicated the presence of two titrata~le groups at PKa values o~
abou~ 5.4 and 7 D 1, respectively, with po~sibly additional pKa's ~10 (initial pH 5.93). An observed molecular weight of about 1555 W25 obtained using fast a~om bombardment mass spectrometry. A tentative empiric 1 70 64C13N7~28 is assigned to factor F
The molecular weight data sug~est that factor F differs from .actor A by he addition of two 1~ su~ar moietLes, an~ an uneven moleculaY weight in~i-cates th~re is no amino sugar present.
The infrared absor2tion spectrum of anti-biotic A41030 factor ~ in KBr pellet is shown in the accompanying drawings as Fig. 6. The following dis-guishable absorption maxima are observed: 3~48-3~26 (stron~, broad), 1653 (strong), 1600 (medi~m), 1504 (strong), 1429 (weak~, 1258 (weak), 1227 (strong), 1136 (strong), 1075 (strons), 1053 (strong~, and 1010 (strons) c~ '.
The ultraviolet abscrption maxima o' A41030 fac~cr F in neutral, acidic, and ~asic methanol:water (1:1) are -secGrded in Table 5 above.
Antibiotic A~1030 fac~or ~ is s~luble in the same solvents as actor ~.
3~

On the basis of the observed physical chemi-cal data, the following structure has been assiglled to A41030 Factor F:
QH

" ~ NHo ! ~ ~ a/ \CI Galactosyl-Galactos~-9/
~ H OH

Antibiotic A41030 factor G is a white solid hav-ing an approximate elemental analysis as follows; 50.02%
carbon, 4.61% hydrogen, 4.74% chlorine, ~ nitrogen, and 30.70% oxygen. Electrometric titration of factor G in 66~ dimethylformamide in water indicated the presence of titratable groups at PKa values of about 5~4 and 7.0 respectively, wi~h possibly additional p~aSs >10~5 ~initial p~ 6.32~. An observed molecular weight of ahout 1684 was obtained using fast atom bombardment mass spectrometry~
The infrared absorption spectrum of antibiotic A41030 factor G in KBr pellet is shown in the accompanyin~
drawings as Fig. 7. The following distinguishable absorp-tion maxima are observed: 3320 ~very broad, strong), 2975 (sharp, weak), 2920 (sharp, weak), 1659 (normal, strong), 1594 (broad~ strong), 1512 (sharp, strong), 1492 (shoulder), 1430 ~sharp, weak), 1386 (broad, weak), 1337 (broad, weak), 1308 (sharp~ weak), 1264 (sharp, weak), 1230 (broad, medium), 1145 (broad, medium), 1077 (sharp, medium), 1062 (sharp, medium), 1014 (sharp, medium), and 846 (broad, medium) cm 1.
The ultraviolet absorption maxima of A41030 factor G in neutral, acidic, and basic methanol:water (1:1) are recorded in Table 5 above.
. An~ibiotic A41030 Eactor G is soluble in the same solvents as is factor A.

~ ,~L

X~5984 -22-Factors A, B, C, D, ~ , and G of the A41030 complex can be. separated and disti~guished from vne another by employins silica-sel thin~layer chromat~g raphy ~TLC) and paper chromatography. Bacillus subtilis was the organism used for the bioautography.
The ratio of movement (Rx) expressed rel~tive to that of A41030 factor A, which was given a value of 1.00, is set forth in Table 6, which Eollows.
TABL~ 6 1~ Rx Solvent System Factor ~ ~
A 1.00 1.00 ~-0 , ~.76 0.7 C Ø6~ 0.4~
D 0.65 0.91 E 0.49 0.63 F . 0.21 0.2$
G 0.21 0025 System A
Paper. : Whatman ~o. ' (untreated).
Solvent: n-Butanol saturat~d with water:methanol (i: 1) .
Svstem 3 Sorbent: Mersk ~arm~tadt-Silica Gel 60 Sol~ent: Ace~onitrile:ethallol:~at~r ~8:1:1..~)~
The hish pexformar.ce liquid chromatography (HPLC) retention ti~s cf A41Q30 factors A through G, inclusive, wer~ det~rmined usins a stainless steel 25~3 X~59~4 -23-column having 10 micron LiChrosor~ P~P-18 as the packing, with a solvent consisting o wa~er:acetonitrile:dibutyl-amine (82:18:0.03M) adjusted to pH 2.5 with phosphoric acid. The solvent was applied at a flow rate of 0.75 S ml./min. The eluate was monitored by UV absorption at 225 nm. The relative retention values, which are the ratio of the retention time for each factor relative to that of A41030 ~actor A, are set orth in Table 7, which follows.

Relative Factor I Cm.Min. Retention A 6.419.2 1.00 B 4.117. 3 0.64 C ~16.2 0.8~
D 3.811.4 0.59 ~ 2.78.1 0.42 F 4.513.S 0.70 G 4.513.5 0.70 Since the several factors of antihiotic A41030 are amphoteric, containing both an amino group and a carboxylic aci~ function, they are capable or formin~ salts with suitable acids and kases. The pharmaceuticallv acceptable sal~s so ~or~led are also part o this invention. "Pharmaceuticaliy-acceptable"
salts are salts which are useful in the chemotherapy of warm-blooded anlmals. Representative and suit-able salts o~ A41030 actors A, 3, C, D, E, F, and G
include those acid addition salts formed by standard X-5984 ~~

r action with ~oth orsanic and inorganic acid.s such as, for ex~mple, sulfuric, phosphorlc, hydrochloric, ~cetic, succinic/ citric, lactic, maleic, f~mariG, palmitic, cholic, pamoic~ mucic, D-glutzmic, d-cam~
phoric, glutaric, glycolic, phthalic, tartaric, lauric, stearicl salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic and li~e acids, as w~ll as salts formed with the caxboxylic acid functiQn with ~uch bases as sodium hydroxide, sodium carbonate, potassium carbo~ate, calcium hydroxide, potassium l~ hydroxide, trimethylamine, am~oni.um hydroxide, di-ethanol~nine, and like bases.
Antibiotic A~1.030 complex and factors are ~ctive against gram positive ~icroorganisms, includins Staphylococcus and tr~ptococcus species~ ~hese an.i-~5 biotics ~l~o show activity for growth promotion and - -improving feed efTlciency ~ poultry, swine, and cat~le.
The activity of the A41030 complex and the individual factors has been demonstrated by a num~er OT
tests which are des~rib~d hereinafter.
~ntibiotic A4103C factors A, B and C have been tested and found to be 2C~ ' ve aaains~ a genus of anaerobic bacteria identified as Propionibacte~ium acnes. The MIC values were determ~ned by the 24-~our ____ agar~dilution method, and are set lorth in Ta~le 8, ~-2~ which follows.

3~

X-~8~ ~5 P~(;AINST PROPIOMIBACT:ERIUM ACNES
Strain of P~IC (ll~./ml. ) P. acnes A B C
44 0.125 ~. 06 0.. 1 25 7g a. 12~ 0. 06 0.1~5 101 0 . 125 0. 06 0. 125 ï03 ~ . 125 0 . 06 0 0 125 104 0 . ~5 0 . ~5 0 O ~5 l~S ~ . 125 0 . 06 0 v 125 1 ~ 6 0 . 1 2 5 0 . 0 6 0 . 1 2 5 107 0 . 06 0. 06 0 ~ 1~5 ~2 0. 06 0. 06 0. 0 5170 <0.0~ <0.03 <0.03 ~176 <0. 03 0. 06 <0. 03 ~1~7 <0. 03 0 . 06 0. 06 5191 0 . 125 0 . 06 0. 125 ~197 _0. 03 0. 06 ~0. 0~
2~ 5226 0. 5 0. 5 0. 125 ~227 <0. 03 0. 06 0. 06 -522~ 1.0 0.5 1.~
522~ 0.~5 0.S

5~4~ 0 . 0~ 0 . 125 0 . 06 Antibiotic A41030 factors A, B, C, D, E, F, and G have been tested and found to be active agai~st a nurnber of anaeroblc bac teria, as reco~ded in Ta3~1P g, which ~ollows, the MIC v21ue~ ',avin~ be~n determined b~
tl~ agar-dîlution method.
3~

WN ~ ~ 1-- ~I t ~ t~ O Ul T~BLE 9 A~,TLVITY OF A4103~ FACTORS AGAIN~ ANAEROBIC BhCTE~IA
MIC ~g.ls~l.) Te~ Organism A B C _ _D _ E F G
Clo~ridIu~ ~fficlle 2994 32 32 16 0.5 0.S 1.0 2C1O6frldlum perfri~2g~ns 81 0.5 0.5 1.0 0.5 1.0 1.0 2C~ r~lu~ septl~ 28 2 4 8 0.25 0.5 1.0 2Eub~cter~u~. aerofaciens 1235>128>128 >128 0.5 0.5 1.0 2~' p~oc~ccus asacc~l~rolyticus L302~0.125CQ.25 ~0.25~0.125 '-0.25 <0.25 1.0 ~eptoc~cc~Y preYotl :L281 ~0.25~0.25 ~0.25 0.25 32 32 2 s PeptostLep~ococcu& ~naerobiu~ 1428~0.25c0.25 ~0.25 0.5 32 3~ C0.5 Peptos~reptococc-~s iutermedius 12641.0 0.5 0.5 1.0 32 1.0 Z
Proplunibacterium acnes 79 <0.25 16 ~0.25 0.25 0.5 1.0 1.0 Bacteroides ~r~g11is 11l. 128 64 .>128 32 S4 32 32 ~"c~eroides fr~gills 1877 32 3~ 16 32 32 32 32 Bacteroide~ ragll Ls 1936B 64 32 32 32 64 32 64 Bac~erGides ~hetaiot~microtl 1438 64 32 64 32 ~4 64 ~4~cteroide~ e1aninogenic~ls l856/28>12~ >128 ~128 >64 ~128 ~128>128B~ct~ro~es ~elan1llogenlcus 2736 4 4 4 0.5 32 1,0 64 P,~cr~roides vulgati~ 1211 32 32 3Z 32 32 32 64 Bac~e~ides corroden~ 1874 64 64 32 32 64 32 32 Fuso~acterium ~ylllblosum 1470 l.a l.Q 1.0 0~5 1.0 1.0 2 ~uso~cteriunlllccrophorum 6~54A 8 8 16~0.l25 0,5 1.0 2 X-598~ 27-The A41030 antibiotic factors A, B, C, D, E, F, and G, are also active against a numbex of strains of Clostridium difricile, as determined by the aga_-dilution methQd. Tne resul~s of the tests are recorded in Table 10, whlch follows.

ACTIVITY OF A41030 FACTORS AGAIl~ST
CLOSTRIDIUM DIFFICILE STRAI~;S

Clostridlum ~IIC (llg. /~1. ) 0dif f lcile A B C D E F G
, _ ~484 1.0 1.0 1.0 c0.25 ().5 1.0 1.0 6890 1.0 1.0 2 0.5 0.5 1.0 1.0 2634 1.0 1.0 2 0.5 1.0 2 1.0 7~ 1.0 0.5 1.0 <0.2j 0.5 l.O 1.0 4 1.0 1.0 2~0.~5 0.5 1.~ 1.0 A-195 1.0 1.0 1.0 <0.25 0.5 1,0 1.0 A-196 1.0 1.0 2 0.5 1.0 2 1.0 ~-27~ 1.0 1.0 2~0.25 0.5 1.0 1.0 ~280 1.0 0.5 1.0 <0.25 0.5 1.0 1.0 A-231 1.0 1.0 2 0.5 1.0 2 1.0 WAL-2112 1.0 1.0 2~0.25 0~5 1.0 i.0 h'AL-3657 1.0 1.0 2<0.25 0.5 l.O 1.0 WAL~42S8 1.0 0.5 1.0 <0.~5 0.5 1.0 1,0 107B 1.0 0.S 1.0 <0.25 0.5 1.0 1.0 ~5lllF 1.0 1.0 2~0.'75 0.; 2 1.0 il53 1.0 1.0 2 i.~ 1.0 2 1.0 3424-SB 1.0 1.0 1.0 0.i 0.5 1.0 1.0 381~i 1.0 1.0 2 0.5 0O5 i.0 1.0 3~50D 1.0 i.0 2 0.; 0.5 1.0 1.0 3~

,.~.. ,.. ......... ,. , . , .... ..... ,, . , ,,, ,, .. , ,,,.. ,.. , ,... , .. ,.. , ............. .... ......... ,...... ~... .
.. . . .. . .... .. ... ... ..... . . . .... .......... ..... ........ ..... . .. .. .. . .... ..... .. . . ...... ... .
. ..... . ...... .. . ~ . . .

f'Pq -X-5984 -28~

The in vitro activity of antibiotic A41030 f~ctors A, B, C, D, E, F, and G as~inst a number of aervbic bacteria has been determirled using a standard agar-diluti~n assay. The results ater reading the end point aftex 24 hours are recoxded in T~ 1e 11, 5 which follows.

~0 3~

........... .................... ... ...... ........................ .......... ............. . ... . . . . . . . . ...

X~5984 -2~-I Lr~ L, .n O _I ~ ~ ~ o c~
A A A ~"
u~ .n r) O O ~ ~/ L'l A A A A

u~
~V~ L"l Ll~ Lt'~ LrlLl--~
~a ~ ~ ~ ~ ~ ~ ~
O O O O O ~ O O _I ~ I 1 0~ CO L~ 0~ 0) CQ
_~ A A A A
r~ In Lr~
A . ~~I ~J ~ ~I L~lLl--l C~C~ ..... .. I
~_~ O O OO O --I O O ~ 1 I LO a ~ v CO ~
t~ ~ l `D
~,) A A
:S
c~ n n ~n u~
~ ;~. . . . I
o o o ~c: o ~ ~ _ ~ ~ ~o ~ ~ I ~ ~ oo ~ 5 c A A A A
E~
.n .n .n .n _ ,~ c~, ~ Lr, ~ Lr~ , 2C~ O C O O O ~-I ~/ Ot7 ~I I I t~ 01~ CO CO
.h r~ r~ r~l ~
~¢ O
2 0 ~ "~, r~r--I LA ~1 r--i LA Lr~
c~ o o ,~ ~ ~ o ~ J c~
o ~
,_~ A A A
~r ¢

';<
O ~ , O ' æ
'7 C
,s._, a u~ o ~: ~ u o ~, ' a ~ _l ~ ~r~ ~ Lt~ ~
- ~ a r~ ~
~') '1~ I ~ O O
~J ~ J r~ O
i r- r _ r~ Z '~
O --~0 ~ U
,, ; , _ ~ U ~ Q
~ J ~ .C~ r-- r~r~_ _ _ _ ~n CC ~ r~ ~r ~ . U u .. ' ~! .L _J ~ J~,1 ~ - ' ' I
Ul U' tl~ tn V~ . U~ ~ L t:

o ~n c u~
- TABTE 11, cont~nued . MIC ~g./m1,) Tes~ O~ganls~ A B _ C D E F G
. K1ebs1ella pn~umoni~e X26 '>128 >128 ~128 128 >l28 >128 64 -' ~1e~ie11a pneumoniae K~E >128 >128 -~128 >128 >128 >12~ >128 ~ebsi~ila pne~ iae X68 . >128 128 ,128 >128 >128 ~128 ,'128 En~ervbacter aero~enes C32 >128 64 >128 >128 >128 >128 >128 En~erobaeter aerogell~s EB17 ;~28 >128 >128 >128 >128 >128 >128 .~ Elltcro~acter ~10ac~e EB5 '1~8 128 ~128 >128 `128 -~128 >128 -Lteru~3cter ~lvacae 265A >L28 128 >128 >128 >128 >128 >128 Sa1monel1a ~yphl X514 >128 >128 >128 >128 >128 >128 >128 0 ~b 5almon~11a ~y~ 1 l335 ~128 >128 :.128 ~128 >128 ~128 ~128 ~selldv.lmonas aeru~.:Lnosa X528 ~l28 ~128 >128 ~12& >128 >12~ >128 ~seli~omGIla~ aerugillo~a X239 ,-128 >128 >128 >128 `-128 >128 >~28 C~
Ps~udumuY~as aeruginosa P~18 . >128 >128 >128 >128 ,'128 >128 >128 Pseudomol~as aeru~ osa Ps72 ~ 128 >128 >128 >128 Serratia ~arcescens X9g >128 128 >128 >128 >128 >128 >I28 Serra~ia m,~cescens SE3 ?128 >128 >128 >128 >128 >128 ~128 Prot~u~ morga~li PR15 `~128 ~128 >128 >128 >128 >12~ >128 Proteus 1~consta~ls PR33 '128 '128 >128 >128 >1~8 >128 >128 Pr~teus rettger1 PR7 ~128 >lZ8 >]28 O ~ O ~ o tn TABLE 11, cont~ l~tled ~IIC (llg . /ml. ) - Test Organlsm ' A B C D E F G
Proteus rettgerl CZ4 >128 >128>12B >128 >128 >128 >lZ8 ~itrobac~ Ç2:eurldii C~l 7 >lZ13128 >12& >128 >128 >128 >128 Bordetella brollc~iseptlca 16 >128 >128 >128 Acinetobact~r calcoacet~cus AC12 -- -- -- ~128 >128 >128 >128 - -- not te~ted 32?~

The acti~ity of antibiotic A41030 complex against a num~er of animal pa1-hogens was determined by a standard in vi~ro antimicrobial broth ~icroti~er test, and the results are set forth in Table 12, which follows.
TABLE 1~
ACTIVITY C)F A41030 COMPLE~ AGAINS
5EVERAL A~I~AL PAT~OGENS
Test Organism MIC ( lJ~ . /ml .
Staphylococcus ~ 1130 ~0.78 -Streptococcus ~ 80 <O.78 Pasteurell~ multocida ~bovi~e) 3.12 Pasteu-ell2 hemolytica 6.25 1~ 3Ordet~11a bron~hiseptlca (S~ritzer) 50.00 Escherichia ~-oli 50,00 Mycoplasma synoviae 50.0~
Mycoplasma hvorhinis 50.00 ~seudomona~ -fish <0.,8 A~romonas liquefaciens 50,00 ~ .
. All of the .~41030 factors tested have sho~m in vi~o antimicrobial activity against experimen~al ba~terial infections. When two doses of test compound were admin~stered subcutaneously to mice in illustrative inf~ctions, the activitv observed is .~easured as an ED50 value ~erfectlve dose in my./kg~ to protect fifty pexcent of the test animals: See liarren Wick, et al , J. Bacteri.ol. 81~ ~J3-235 ~1361)]. ThP ED values observed ~o~ A4l030 factors ~, B, Cl D, E, a~d F ar~
given in Table 1~, which rollows.

X~5984 -33-Staph. aureus S. pyogcnes S. pneumoniae Antibiotic E _ ED-50 ED-50 A~1030A 1.4 2.8 1.~8 A41030B ~0~43 1.4 1.4 ~1030C <0~43 10.4 ~.7 A41030D 0.339 3.24 2.21 A41030E <0.31 3.54 3.11 A41030F <0.31 >~.0 ~5.0 1~
The acute toxtcit~ of each of the antibiotic A41030 factors A, B, and C, has been determi.ned in mice and has been found to be ~300 mg./k~.
The LD50 OL each of the anti~iotic,A41030 ~' factors Al B, and C, has been determined in mice as belng ~300 mg./kg.
The _ vivo oxal activity of each oE the antibiotic A41030 factors A, B, and C, as determined against S. pyogenes in mice, is >300 mg./kg. X ~O
In one of its aspects this inventi.on provides a method for treatin~ infections in a warm-blooded animal which comprises administerin~ to said anirnal a c~emotherapeutically-effective amount; for example, of between about 25 mg~ and about 2,0~0 mg~ of the ~41030 ~-antibiotic comple~{, or factor thereof, or a pharma-ceutlcally-acceptable, salt thereor.
Factor A, or a ~harmaceutically-acceptable salt thereof, can be pre~erably used in the trea-~ment of in~ections in mant but in ~eneral, the complex and oth~r ~actors and salts thereof are best suited for use 3~ in the -treatment o~ in~ections in other ~arm~blooded an iIn~ 1 S .

~6 X-5984 ~34~

In the treatment of infections in man the antibiotic factor, preferably factor A, can be admin-istered by the parenteral route, e.g., by i.m. injec-tion, or i.v. infusion. For injection, ~he antibiotic S or a pharmaceutically-acceptable salt thereof is dis-solved in a physiologically a~ceptable diluent at the desired concentration and administered. Suitable diluents include for examp~e, Water for-Injection, 0.9%
saline, 5~ dextrose, Ringer's sollltion~ or oth2r co~-monly employed diluent. For administration by i.v.
infusion, the antibiotic or sa:Lt thereof can be made up in a physiological fluid or dilu-te nutrient at a suit~
able concentration; for example, at a concentration between about 5% and about 10~, an~ slowly infused with 1~ the fluid. Aiternatively, the antibiotic may be ad,~in istered by the "pigyy-bacX" method.
The individllal factors, combinatio~s of the factors, or the whole comple~ of factors and the pharma-ceutically~accepta~le salts thereof ~an be made up in dosage unit formulations in hermetically sealed vials, sterile, rubber-stoppered vials, or in plas-tic pouches.
Such unit dosage ~orms can contain excipier,ts such as antio~idants, so1ubilizlng agents, dispersing agents, buffers, and the like. One such ~osage unit forinula- ~
tion comprises 100 mg. of factor ~, or a pharmaceu-tically-acceptable salt thereo J in a rubber (hutyl rubber) stoppered vial. Another dosage unit formula-tlon compri.ses 250 mg. of ~actor A, or a salt thereof, in a ster:Lle, sealed vial. For i.v. infusion a dosage unit formulation of this invention comprises 5 g. of 3 factor A, or a pharmaceutically-acceptable salt thereof, in a plastic pouch.

.

2~

X 5~8~ ~35~

When ~41030 complex or an A41030 factor is used as an antibacterial agent, i~ may be administered either orally or parenterally. As will be appreciated by those skilled in th~ art, the A41030 complex or ~actor -~s commonly administered together with a pharma-ceutically-acceptable carri~x or diluent~ The dosage of A41030 complex or factor wiil depend upon a variety of considerations, such as, for example, the nature and severit~ of th2 particular infection to be treated.
Thos~ skilled in the art will recognize that appro-priate dosage ranges and/or dGsage units for admin-istration may be determined by co~sidering the MTC and ED5~ values and toxicity data herein provided, together with factors ~such as ~he patient or host and the infectir.g or~anis~..
The A~1030 antibiotics ~re useful n~er alia for suppressing 'he srowth or Staphylococcus, .Stre?to-coccus and Propioni~acterium acnes or~anisms, and the antibiotics could therefore be used, for e~ample, in the treatment of acne. The A41030 individual ~actors, o~ mixtures thereof in the puri~ied state, can be formulated in phaxmaceuticall~-acceptable diluents suc~
as isopropyl alcohol Cor application to the skin. 5uch solutions can be ~ade up with antibiotic concentrations or rrom about 1 to about 15 percent weight pe- volume.
~5 Altern2tivelv, khe anti~iotics can ~e made up intc creams or lo~ions for applicatlon to the skin.
The A41030 antibiotics are also us~ful for suppressing the growth of Clos'ridium difficile organ~sms, which cause Pseudomembranous colitis in 3~ the intestine. Thf' A4103Q ~ndividual factor~ or mix-....................................... ...... . .. ....... ............ .. .......................... ................. ............ ... ..

329~

X-59~4 -36-tures thereof could be used in the treatment o Pseudo-membranous colitis by the oral administration of an effective dose of said antibiotics or a pharmaceutically-acceptable, non-toxic salt thereof, prepared in a pharmaceutically-acceptable dosa~e form. ~or such use the antibiotic can be administered in gelatin ca~sules or in liquid suspension.
The antibiotics of this invention also can be used in veterira.ry medicine in the ~ eatment of infec-1~ tious diseases in domestic and farm anlmals. They areuseful also in animal husbandry, e.g., in enhanclng the growth of b~er cattle and other ruminants. An espe-cially valuable use for the antibiotics of this inven-tion resides ln their ability to increase the produc~
1~ tion of milk in ~airy cattle. These uses are further aspect~ of this in~rention which are desc:ribed in more detail in the following paragraphs.
The A4103n complex has s~own activity against infectious canine hepatitis virus in vitro at 40 mcg./ml. The A41030 complex has also shown activi-tv ln vitro against pseudorabies at 20 mcg~/ml; and ~1030 _ factor A has shown activity in vitro against pseudo-rabie.s at 20 mcg./ml.
~he A41030 co~lpleY~ can be produced b~ cultur- ~-ing the previollsly unclescribed mic~oor~nism Strepto~
m~ virginiae NRRL 1~525, or an A41030-producing mutant or variant thereof, in a culture medium contain~
i~g assimilable sources of carbon, nitrogen, and inor-ganic salcs, under submerged aerobic fermentatlon conditions until a substantial le~el of antibiotlc activity is produced. Most of the antibiotic ac~ivi~y X-598~ -37~

is generally found in the broth, while minor amounts of antibiotic activity may be associa~ed with the mycelia.
The ~41030 complex is most readily separated from the fexmentation mixture by removal of the mycelia, i.e., the biomass, by filtration. The mycelia are generally discarded. The antiblotic complex is then isolated from the liltered Cermentation broth pre~erably by column chromatography~ over a suitable adsorbent using, for example, methanol:~.ater (1:1) as the elutin~ agent.
1~ ~uitable adsorbents include car~on, alumina, anion and cation exchange resins, silica gel, po]y-amide, carbo~ymethylcelluloses, highly porous copoly-mers of stvrene and divinylbenzene such as Diaion HP-20, the ~mberlite XAD resins, and the Duol~te resins such as E~-865 ~r.d the ~ike, as well as Sephade~ resins, tne hydrophilic, insoluble, molecular-sieve chromato~
graphic mediums made by cross-linkin~ de~tran, ancl also TSK Gels. The Diaion resins are a procluct of Mitsu-bishi Chemical Industries, Limited, Tok~o, Japan. Tne Rmberlite XAD resins are produced by Rohm and E~aas, Philadelphia, Pennsylvaniar The Duolite resins are products of ~iamond Shamrock, ~edwood City, Caliornia.
Sephadex resins are rnanufactured by Pharmacia Fine Chemicals A~, ~ppsala, Sweden. The TS~ Gels are avail- --able frcm E. Merc~c, Darmstadt, and from Bio-Rad, 2200 ~right Ave., Richmond, California, 94804.
The A41030 anti~i~tic comple~ can be further purified and separa~ed into lts individual ractors by chromatocJraphic techni~ues.
A number of diC_Qrent media may be used with 3 Streptom~ces ~Jirginiae NRRL 1~525, to produce ~he X-5g~ -38-A41~30 complex. For economy in production, opt~al yield, and ease of product isol.ation, however, certain ::ulture media are preferred. These media should contain assimilable sources O! caxbon, nitrogen, and i~organic salt~. Suitable carbon sources inolude dextrin, starch, mannose, slycerol, and cottonseed oil.
Optim~m levels of carbon sources are ~rom about 2 to about 3 percent by weight.
Preferred ni~rogen sources include soyhean grits, soybean flour, peanut meal, rish meal, meat 1~ peptone, and pork blood meal.
Essential trace elements necessary for th~
growth and de~elopment of the organism may occur as impurities in other corlstituPnts of the media in a~ounts su~ficient to meet the growth a,nd biosynthetic requir2ments of the organism. However, it may be beneflcial to incorporate in the culture media addition~l soluble nutrient inor~anic salts c~pable of yielains sodium~ potassium, magne~ium, calcium, ~mmoni~m, chloride, carbonate, phosphate, sulfate, nitrate and 2~ like ions.
Addition to the 'ermentatior. medium cf Tween 80 ~oily liquid ~ol~oxyethylene sor~ltan mono-oleate, a product of ICI Americas, Inc.~ Wilmington, Del~), at ~ level of 2-4% se-ves to increase the yield ~5 by about 300%. ~owe~err di~ficulty is expexie~ced in isol~ting the A41030 antibiotic u~der these conditions.
Althou h sm~ll quantities o. the A41030 antibiotic may be ~btair.ed by shake-rlask culture, sub~erged aerobic fe~mentation in tan~s is preferred 30 for produ~lng substan~ial quantitles of the A41G30 .............. ..... .. . .. . ........ ..... .. . . .. .............. ... .. . .. . .. ... ...... .. ... . .. ......... ............... . .. . .
.. . :.. ...............................................................................................................................................................................
....

X-598~ -3g-antibiotic. For tank fermentation, it is preerable to use a vegetative inoculum. The vegetative inoculum is prepaxed by inoculatins a smal.l volume o~ culture medium with the spore fonm, or mycelial fragments, to obtain a fresh, actively growi:ng culture or the organism.
The vegetative inoculum is then transerre~ to ~ la~ger tan~ where, after a suita~le incubation time, the A~103~ antibiotic is produced in opt~l.~l yield~
An alternate method of providing inoculum for the vegetative medi~ consists of ~ubstituring a 1 lyophilized pellet for the a~ueous spore suspension.
Lyophili7.ed pellets are prepared in a ~,anner known in the art. Preparation of the spore s~spension for lyophilization ls similar to the preparation of the a~ueous spore suspension, except that sterile calf serum is su~stitu.ed _or sterile distilled water.
The ~41030~producing organism can be grown over a broad temperature range of from about 10 to about 34Co Optimum production of A41030 anti~iotic complex appears to occur at a temperature of about 30C.
As is cus.omary in aerob.c submer~d cul~ure processes, sterile air is di~pe~sed through the culture medium. For efficient growth of the organism, ~he volume of the air used in tan~ production ls in *he ~5 ra~ge o~ rrom a~out 0.1 to about 0.5 volumes of air per volume of cultllre medi~m per minute (v/v,tm), with fr~m about 100 to abou. 300 R~M agitation. An optimum rate in a 1~5-liter ~essel containins 100 liters of ferme~.
tatio~ medium is about C~5 v/v~m, with agitation 3~ pr~vided by an impeller _ot~ting a' a~vut ~00 RPM~

.. , ., . ;..... .......... ~ .. .................

X-5984 _~o_ Antibiotic activity i.s generally present after about 48 hours and remains pres2nt for at l~ast 144 hours during the fermentation p~riod. Peak anti-biotic production occurs ~ rom about 9~ houxs to about 120 hours f rmentation time.
Production of the A41030 an~ibiotic c~n be monitored during the fermentation by either agax dif-fusion using B. subtilis~or a turbidimetric me-thod using Staphylococcus aureus ATCC 9144.
In OL der to illustrate more fully the opera-tion of this lnventi~n, the followin~ ~xamples are provided.
Example 1 Prepar~tion of First Stage Inoculum The following m~di~l was prepared for use in the ag~r slant cul~ure o~ Streptomyces virginiae NR.~L 12525: , In~redient Amount (g.~L.) ~0 Dextrinl lQ.0 YPast extract 1.0 En~me-hydrol~zed casein 2.0 ~e~f ~xtrac~
CoC1 ~6H O 0.01 ~gar ~eionixed water q.s. ~o 1 liter Matheson Colem~n ~ ~ell, No.rwood, Ohio ~5212 N~æ-~mine ~(Hu~Xo She.~fie7d Chemical Co., Memphis, 3~ Tenn.~O

5 9 ~

The p}l of the medium as preparPd wa~ , and was ad justed to 7 . 3 usirlg 5 N aqueous sodium hydrc: xide befor2 autoclaving. After autoclav.ing, the pH of the medium was 6 . 9 .
Spores of Streptomyc~.s virginiae NRRL 12525 were inoculated on an agar slant made up of the above-identif ied ingredients, and th~ thus-inoculated slan~
W2S incubated for abou-t six days at a temperature of about .30 C . The makure slant culture was then covered with st~rile distilled water ar~d scraped wi .h a sterile tool to loosen the spores and ~he mycelium~ One mi~liliter of the resulting spore suspension was used to inocula~e 50 ml. of vegetative medium. An alternate rnethod of providing inoculum for the vegetati~e medium consisted of substituting a lyophilized pellet for t~e l~ aqueous spore suspension. The composition or the vegetative medlum was as ~ollows:
Insredient Amount (g . /L. ) Glucos~ 20. 0 2~ Soybean grits (or soybean flour) 15.0 Cor~ steep liq~o~ lO.0 C2C 3 2~ 0 Tap water q.s. to l li~er The unadjusted p~ of the medium was 5.S, ~-which was adjus~ecl to pH 6.5 with 5 ~ aqueo~ls sodium hydroxid2 b~fore autocla~in~. The p~; o. the mediurn al e~ autcclaving w~s 7. 0 .
The veg&tatlve inoculum was incubated in a 250 ml, wide mouth Erlenmeyer flask corltaining ~0 ml.
3~

.. . . .. ., . .. . .. . . . .. .. .. . . . . . - . -X~59~4 -42-of mediunt at about 30C. for about 48 hours on a shaker rotating through an arc 2 inches in liameter ,at 25~ RPM.
Thi~ incubated snedi~ s used either to inoculate small fermenters (the lnoculum being approximately 1% per vol~te of fermenter medium) or to inoculate a secon~
5 stage medium having thP sante s~omposition as the vege-tative mediuIst or the pxoduction of ~ larser volume of culture .
Ferme.ntat i on of A~1030.4 1~ Fifty milliliters o. a production medium wa~
inoculated with 1% (0. 5 ml. ) of the incubated vegeta-tive medium from above. The production m~edium had the .~ollowing compositlon:
Ingredien~ ~P.mount (c~
Potato dex~rin 3o.
Soybean grits 6. 0 ~C2~P04 1. 0 FeS04 7H20 M~S04 ~ 7H20 1. 0 NaN03 1 ~ 0 C~C03 Deioni7ed water q. s. to 1 liter The ~HP04 was dissolv~d in water, t~e solutton s terilized se~arately, an~ the requisite amcunt of the solution added to the other in~edients o~ the medium th~t had been autc)claved.
The inoc~tlated fer~ent~tion mQdiu~n, 50 ml., was ~ncubated in a 25C-ml. ErlenmPyer flask at ~bout 3~ 30~{~. for about 4 5 days on a shaker rot~tting through aTt arc 2 inches in diametex at 250 ~M~

- , . , . , - ,-,, . , , ~ . . . . . . ~ .. . .. ... ......... ....... .........

-The Streptomyces virginiae N~R~ 12525 was also incllbated in a fermentation carrie~ out on a larger scale in 165~1iter and 1600 gallon tanks using the production medium described immediately hereinabove.
The inoculated production medium was allowed to ferment in a 165-liter fermentation tank containing 100 liters of medium for about 210 hours (8075 daysj at a temperature of about 32C. The fermenta~ion medium was aerated with sterile air at a rate of 0025 v/v/m and was stirred with conYentional agitators at abvut ~00 RPM.

Example 2 Separation of A41030 ~ntibiotics Whole fermentation broth (421S liters), obtained as described in ~xample 1, was filtered using a filter aid (Hyflo Supercel~, a diatomaceous earth, Johns-Manville Products Corporation) in a filter press. The filtered broth was applied to a column containing 100 L. of Diaion~ HP~20 ~a highly porous styrene-divinylbenzene copolymer in bead form, Mitsubishi Chemical Industries, Limited, Tokyo, Japan~ at a flow rate of 4 L /min. The column was washed successively with 300 L~ of water and 1000 L. of methanol:water (1-3) at a rate of 4 L./min.
Elution wa~ performed with methanol:water (1:1) at the rate of 6 L./min., collecting 100-L. fractions. Each fraction was analyæed Eor biological activity. The bio-assay was performed by a paper disc assay on agar plates seeded with ~acillus subtilis Fraction 1 was discarded.
Fractions 2-15, inclusive, were combined, concentrated under reduced pressure~ and the concentrate lypohilized to give 200 g. of crude antibiotic complex.

A portion of this complex, 110 g., was dissolved in 5 L~ of methanol:water (1:1), by adjustment to pH 10 with aqueous sodium hydroxide, and the mixture was fil-tered. The filtrate was applied at 50 ml./min. to a 30-L.
column (0~2 x 1 m.~ of coarse Sephadex G-50~ (a hydro-philic, insoluble, molecular-sieve chromatographic medium, made by cross linking dextran, and sold by Pharmacia Fine Chemicals, Piscataway~ NJ 08854), previously equilibrated with methanol:water (1:1~. The column was eluted with methanol:water (1:1~ at 50 ml./min~, collecting 3-L.
fractions. Fractions 1-12~ inclusiv~, were discarded.
Fractions 13-24, inclusive~ which contained activity against B. subtilis, were combined, concentrated under reduced pressure, and lyophilized to give 35.7 g. of the A41030 antibiotic compl x.

Example 3 Isolation of A41030 Factor A
An 8 g. portion of the A41030 complex from Example 2 was dissolved in 200 ml. of a solvent consist-ing oE water:acetonitrile:sodium chloride (84:16:2 g./L.
and filtered. The filtrate was applied to a stainless steel column (9 x 100 cm~) packed with 4 L. of 10-20 micron LP-l/C18 reversed-phase silica gel which was prepared in our laboratories by a special procedure described in Examples 6 and 7 of U~S. Patent No.
4,299,763. The column was part o~ a Chromatospa ~
Prep-lO0 unit (Jobin Yvon, 16-18 Rue du Canal 91160 Lon~jumeau~ France). The column was eluted at 60 .~

9~

( X~5984 -45-ml./min. with water:acetonitrile:sodium chloride ~84:16:2 g./L.) collecting 480-ml. fractions. The eluate was monitored a~ 254 nm using an ISC ~lodel UA-5 UV monitor wi~h a Type 6 optical unit (Instrumen-tation Specialties Co., Lincoln, NE 68505~. Selectedfractions were analyzed for the presence of Factor A
by analytical high performance liquid ~hromatography (HPLC) cn a 4.6 x 250 mm. stainless steel column packed in our laboratories with 10 micron LP-1/C18 which was prepared in our laboratories by the special procedure described above. The sample was applied with a Rheodyne Model 7120 injection valve (Rheodyne Inc., Berkeley, CA
94710). The solvent, consisting of water:acetonitrile:
sodium acetate (81:19:0.03M) adjusted to pH 6 wi~h glacial acetic acid~ was supplied at 1 ml~/min. (1200 psi) by a .~ilton Roy Duplex Minipump (Laboratory Data Control, Division of Milton Roy Co., Rivera Beach, F1 3340~). Factor A was detected at 254 nm using an ISCO
Model UA-5 UV detector. Fractions 1-51~ inclusive, were discarded. Fractions 52-79, inclusive, rich in factor A were combined and ccncentrated under reduced pressure to a volume of 500 ml. The concentrate was adjusted to pH 8.2 with aqueous sodium hydro~ide and filtered. The filtrate was applied at 15 ml./min. to 100 ml. of Diaion HP 20 resin in a column ~2.8 x ~2 c:n.) previously equilibrated wi~h water. The column was washed with water (400 ml. ad~usted to pH 2.5 with formic acid) until no chloride was detected in the wash ny ~ecipitation as silver chloride. Elution was ~erformed with water:ace~onitrile (3:2) at 15 ml.~min., -~6-collecting 1 L. fractions~ F~ractions were analyzed for activity against B. subtilis~ Crystalline factor A, which formed in fraction 2 upon refrigeration, was recovered by filtrati~n (38g.6 mg.). Fraction 1 and the filtrate from fraction 2 were each concentrated under reduced pressure and lyophilized to give 731.8 mg. and 514 mg. of factor A, respectively~

Example 4 Isolation of A41030 Factor B

A 1.0 g. portion of the A41030 complex was dissolved in 35 ml. of a solvent consisting of water:
lS acetonitrile:sodium chloride (85015:2 g./L.) and the solution was applied to a 4.7 x 45 cm. Michel-Miller high-performance-low-pressure-liquid-chromatographv (HPLPLC) glass column (Ace Glass, Inc., Vineland, NJ
08360) packed in our laboratories with 25-40 micron LiChroprep~ RP-18 [hydrocarbon phase (CI8) chemi-cally bonded to silica gel, from MC/B Manufacturing Chemists, Inc., Cincinnati, OH~. An FMI valveless piston pump (Fluid Metering Inc., Oyster Bay, NY 11771) was used to elute the column at 21 ml./min. (100 psi~
25 with the same solvent combination used for sample dis-solution, collecting 21~ml. fractions. The eluate was monitored at 280 nm using an ISCO Model UA-5 W detector.
Fractions 1~183, inclusive, were discarded~ Fractions 184-245, inclusive, rich in factor B, were combined and concentrated under reduced pressure to 25 ml. Concen-trates from seven similar purifications were combined, dilllted to 1~4 L. with water, and applied at ~-10 ml./min.
to 100 ml. of ~iaion HP-20 resin in a column, ,, ., 2~

X 5 9 8 ~I r 4 7 _ previously equilibrated with wate7- l~he column w2s washed with water (600 ml.~ untll no ch}oride was detected in the wash by precipitation as silvex chloride~
Elution was perfo~med with water-methanol (1:1) at 8-10 ml~/min., collecting 300 ml. fractions. Fractions were analyzed for activi~y against B. subtilis. Frac tions 1-5 were combined, concentrated under reduced pressure, and lyophilized to give 523 mgO of crude factox B.
A 550 mg. portion of two combin~d crude prep-arations of factor B was dissolved in 10 ml. sf ~
solvent consisting of water:acetoni~xile:aibutylamine (75:25:0.03M, which solvent had been ad~usted to pH 7O8 with phosphoric acid) ~y addition of tetrabutylammonium hydr~Qxide until solution had been accomplished. The solution was applied to a ~.8 x ;9 cm. Michel Miller HPLPLC glass column packed wi,h 25-40 micron LiChroprep RP-8 ~hydrocarbon phase (C8) chemically bonded to silica gel, from .~C/B Manufacturing Chemis~s, Inc., Cincinnati, OH]~ Using an FMI pump, the col~mn was eluted at S ml./min. (35 psi) with the same solvent combinatio7l used for sample dissolution. The eluate was monitored at 254 r~ using 2n ISCO Model UA~5 W
detector. Selected 27-ml. fractions were 2n21~zed for the presence o~ factor 3 by an21ytical HPLC ~n a 4.6 x 250 m~. sta~ less stee~ column packed with 10 mi crcn LiChrosorb~ P-18 (a commercially available, reversed-phase silica gel) manufactured by E. Mexck, Da~ms~ad.l Gexman~). The sampl~ was applied usin~ a hheodyne Model ,120 injectiQn valve. The solvent, con-sisting o~ wate~:acetonit~ile:dibutvlamine (8~ :0.Q~)a~usted to pH 2.5 with phosphoric acid, was supplied .......... . ............. . .... .. . . . . . ............ ... ..

- X-599~ -98~-at 1 ml.~min. (750 psi) by a Constametric III pump (LDC-Laboxatory Data Contro~, Division of Milton Roy Co., ~iviera Beach, FL 33404~. Factor B W2S detected at 225 nm using ~n LDC Spectro Monitor III variable wavelength W detector. The portion of the RP~
column elua~e from 399-1296 ml~, rich in factor B, was concentrated to a volume of 700 ml. The concen-trate was diluted to a volume of 500 ml., adjusted to pH 2.0 with phosphoric acid, and sodium chloride (1 mg./ml.) was added as an ionic marker. This solution was applied at 20 ml./min. to 100 ml~ of Diaion HP-20 resin in a column (2.B x ~2 cm.), previo~sly equilibrated with water. The column was washed with water ~500 ml.) adjus~ed to ~H 2~5 with aqueous formic acid, until no chloride W2S detecte~ in ~he wash by,precipitation as silver chloride. The column ~as then eluted with 1 L.
of water:acetonitrile (6:4) at 30 ml./min. The eluate was concen~rated under reduced pressure and lyophilized to give 295.6 mg. of crude factor B.
A 285 mg. portion of this preparation was dissolved in 30 ml. dimethylfo~mamide:water (4:6) by heatins, cooled to room temperature, and r~frlgerated, resulting ir. precipitation of f2ctor B. The precip-itate was recovered by filtration, washed with acetone, ~nd dxied l~nder vacu~m, to y~eld 84 ms. of actor B.

3~

~,.

~49~

Example 5 Isolation of A41030 Factor C

A 9.0 g. portion of the A41030 complex was dissolved in 200 ml. of a solvent consisting of watero ~acetonitrile.sodium chloride (83:17:2 gO/L.) and the solution was filtered. The filtrate was applied to an 8 x 100 cmO stainless steel column packed with 4 Lo of 10-20 micron LP-l/Cl~ reversed-phase silica gel which was prepared in our laboratories by the special proce-dure described in Example 3. The column, part of a Chromatospac Prep~100 unit, was eluted at 60 ml./min.
with the same solvent combination used Eor sample dis-solution, and 480-ml. fractions were collected. The eluate was monitored at 254 nm using an ISCO Model UA-5 UV detectorO Selected fractions were analyzed for the presence of factor C by analytical HPLPLC on an 0 8 x 30 cm. Michel-Miller glass column packed in our laboratories with 25-40 micron LiChroprep RP-8. The solventg water:
acetonitrile:sodium chloride ~84:16:2 g./L.~, was supplied at 4 ml./min. by an FMI pump~ Factor C was detected at 254 nm using an ISCO Model UA-5 W detector Fractions 1-27, inclusive, were discarded. Fractions 28 52, in-clusive, rich in factor C, were combined and concentrated under reduced pressure to a volume of 500 ml.
Concentrates from two similar purifications were combined, filtered, and applied at 10 ml./min. to 100 ml.
of Diaion ~IP-20 resin in a column (2.8 x 22 cm.), pre-viously equilibrated with water. The column was washed with water ~2 L~) until no chloride was detected ~9~
X-59S4 -50~
l' .

in the wash by precipitation as silver chloride.
Elution was perfo~med with 1 ];. of ~ater:acetonitrile (6:4) at 15 ml~/min. The eluate was concentrated under reduced pressure and lyophili.zed to give 2.75 g. of a facto~ C~enriched mixture of factors~ A 1.~5 g. portion of this mixture was dissolved in 25 ml. of a solvent consistin~ of water:acetonitrile:dibu~ylamine (80:20:0.03M
which solvent had been adjusted to pH 7.8 with phosphsric acid) b~ addition of tetrabutylammonium hydroxide until solution had ~een accomplished. The sample was applied to a 2.8 x 59 cm. Michel-Miller glass column packe~
with 25-40 micron LiChroprep RP-8 and the column was elu~ed at 4 ml./min./ using an FMI p~mp, with the same ~olvent combination used for sample dissoiution. ~he eluate was monitored at 254 nm using an ISCO Model UA-5 W de~ector. Selected 2~-ml. fractions were analy2ed for the presence of factor C by a~alytical HPLC on a 4.6 x 15~ mm~ stainless steel colu~n ~acked in our labora~ories with 10 micron Nucleosil~C18 (a commercialiy available, re~e.sed-phase siLica gel, manufactuxed by Rainin Instrument Co., Inc., Wob~rn, 01801). The sample was applied using a Rheodyne Modei 7120 injection valve. The solvent, consisting of water:acetonitrile:sodi~m a~etate (81:19:2 s./L.) adjusted to pH 6 ~ith glacia} acetic acid, was supplied r~
2S at 1 ml./min. by a Milton ~oy Duplex .~inipump. ~actor C
~as de ected at 225 nm using an ISCO Model 1800 ~ariable wavelength W detector. The portion of the eluate from 4~2-S~1 L., rich in factor C, was concen r~ted under red~ced pressure to a volumo of 500 ml.

~;,, ~6~
~-5~4 ~51-Concentrates from three similar purifi~a~io~s were combined and dissolved by addition of phosphoric acid to p~ 1.7. Sodium chlori.de ~1 mg./ml.) was added as an ionic maxker. The s~mple was applled ~t 20 ml./min.
to 100 ml. of Diaion HP-20 resin in a column (~.8 x 2~ cm.), previously equilibrated with water. The column was washed with aqueous formic acid sf p~ 2~5 (300 ml.), until no chloride was detected in the wash by precip~
itation as silver chloride. The column was eluted with 1 L~ of watex:acetonitrile (6:4) at 30 ml . /min . The eluate was collected, concentrated under reduced pxessure, and lyophilized to give 0.37 g. of par~iallv purified factor C. This preparation was ~issolved in 20 ml. of a solvent consisting of water:acetonitrile:
dibutylamine (30:20:0.03~.t which solvent had been adjusted to pH 7.8 with phosphoric acid) by addition o tetrabutyl~monium hydroxide until solution had occurred.
T~e sample was ch~omatographed Oll 25-40 micron LiChro~rep P~-3 in a 2.8 x 5~ cm. Michel-Miller glass column, as previously described. The portion of the elual~ from 2.45 3~20 L. was concentrated under reduced pressure to a volume of 500 ml.
Concentrates fxom two similar purifications were combined and desalted on a col~mn containing Dialon HP-20 resin in the fashion previously described. ~^
The elua~e was concentrated under ~educed p~essure and lyophili~ed to give 6~ mg~ of ~actor C. A 673 mg.
~ortion o this preQaration ~as dissolved in 60 ml.
water:acetonitrile ~6:4) by heating. The solution was ooaled and factor C pr2cipitated upon ref-igeration.
The precipitate ~as r~covered by filtration, washed with acetone~ an~ ~ried und2r ~2cu~m to sive 428 mg. of f aotcx C.

......... . ....... ... ......... .. . ... .. ..... .. ........... ..... ... ....... . ... .... .. .............................. . . ... ....
.... . . . . . . . ........... . .

~3~
~-5984 -52-Example 6 Isolation o, A41030 Factor D
A 5.0 ~. portion of the A~1030 complex was dissolved în 200 ml. of a solvent consisting o~ water:
acetonitrile:sodium chlorid2 (83-17:2 gO/L ) and t~.e solution filtered, The fil~rate was applied to an 8 ~ 100 cm~ stainless steel column packed with 4 L. of 10-20 micron LP-l/Cl~ reversed-~hase sll~ca gel which was prepared in our laboratories by the special pro-1~ cedure de~cribed in Example 3. The colurmn, part of a Chroma~ospac Pre~-100 u~it, was eluted at 60 ml./min., with the same solvent combination used ror sampl~
~dissolu~ion, and 480-ml. fractions wexe collected. The eluate was monito-ed at 2~4 nm using an ISCO Model Uh-5 UV detector. ~lec`ed fra~tions were znaly~ed for the presence of Factor D by analytical HPLPLC on an o. a x 30 cm. Mi~hei-Miller glass column packed i~ our l.abora~sries witn 25-40 micron LiChroprep ~P-8~ .he solvent, water:acetoni-trlle:sodiu~ chloride (84:16:2 g./L )~
was supplied at 4 ml./min. ~Ising an ~MI pump, ~actor was detected at 254 nm using an ISCO Model UA-5 UV
detector. Fractions 1-34, inclusiv2, were ~i~car Fractions 35~53, i~clusive, rich in factor D, were combined and concentrated under reduced pressure to a volume of a~ou~ 500 ml.
Concentrates from two similar purifications were com~ined, dilut~d ~o 3 ~. with water, and applied at ~-10 ml./min. to 100 ml. o, Diaion HP-20 resin in a solumn (~ 2~ cm.), ~eviouciy equilibrated with 3~ water. The colu~n was washed with water ~3C0 ml.) -53~

until no chloride was detected in the wash by precipita-tion as silver chloride. Elution was performed with 1 L.
of a solvent consisting of water:acetonitrile (6:4~ at ~ 10 ml./min, The eluate was concentrated under reduced pressure and lyophilized to give 2.33 g, of a factor D-enriched mixture of factors~
A 1.15 g. portion of this mixture was dissolved in 25 ml~ of a solvent consistin~ of water:acetonitrile:di-butylamine ~80:20:0.03M, which solvent had been adjusted to pH 7.8 with phosphoric acid) by addition of tetrabutyl-ammonium hydroxide until solution occurred, The sample was applied to a 2.8 x 59 cm. Michel-Miller glass column packed with 25-40 micron LiChroprep R~-8, and the colwmn was eluted at 5 ml./min~, using an FMI pump, with the same solvent combination used for sample dissolution~ The eluate was monitored at 254 nm using an ISCO Model UA-5 W
detector. Selected 25 mlO fractions were analyzed for the presence of factor D by analytical HPLC on a 4.6 x 25 mm.
stainless steel column packed with 10 micron LiChrosorb RP-18 (a commerically available, reversed-phase silica gel, manufactured by E. Merck, Darmstadt, Germany). The sample was applied using a Rheodyne Model 7120 injection valve. The solvent, consisting of water:acetonitrile:di-butylamine (80:~QoO~03M) adjusted to pH 2.5 with phosphoric acid, was supplied at 0.75 ml./min. using a Milton Roy Duplex Minipump. Factor D was detected a~ 225 nm using an ISCO Model 1800 variable wavelength W detector~ The portion of the eluate from 2~6-3.4 L.~ rich in factor D, was concentrated under reduced pressure to a volume of 300 ~1.

'~.

;P;r3~
X-S984 ~5~

Concentrates from three similar purifîcations were combined and di~solved by addition ~f phosphoric acid to pH 7.7 G Sodium chloride (1 mg./ml.) was added as an ionic anarker. The s~mple was applied at 20 ml.fmin.
to 100 mlO of Diaion HP-20 resin in a column (2.8 22 cm.), pre~iou~ly equilibrated with water~ The column was washed with water (30C ml.) adjusted to pH
2.5 with aqueous formic acid, until no chloride was detected in the wash by precipitation as sil~er chlor de.
The col~ln was eluted with 1 L. of water: acetonitrilP
1~ ~6:4) at 30 ml./min. The eluate was concentrated under reduced pressure and lyophilized to give 0.~3 g~ o.
partially purified factor D. This prepa~ation was dissolved in 15 ml. of a solvent consisting OL wa~er:
acetohitrile.diDutylamine.~80:20:0.03M, which solvent ~5 had been adjusted to p~i 7.8.with phosphoric acid) by aG~ition o~ tetxabutyla~monium hydroxide until solutior.
occurred. The solution was chromatogrAphed on 2S-40 micxon LiChroprep ~P-8 in a 2.8 x 5g cm. Michel~Miller glass column, in ~he manner previously descri~ec~ The portion of the eluate from ~.5-3.0 L. was concen~rated ~nder reduced pressure to a vol~me o~ a~out ~00 ml.
This corAce~trate w.~s desalted on a colu~n conta.ini~g Diaion HP-20 resin in the fashion previously described.
The ~luate was concentrate~ under reduced pressure and lyophilized ~o give 193 mg. of partially~purified factor D.
A 259 ms~ portion or tWO combined partially ~urified factor D preparations was dissolved in ~ ml.
of a soltJent consisting of watex:acetonitrile:dibasi~ -sodi~m phosphate (82~18:0.03~1, which olvent had b~en adj~s~d to pH 7.8 with phospho~ic acid) and adjusted - .. . .. . .. .. . . - . . .. . .. ... ; .. - . - . . .. ...

X-598~ ~55~

to pH 10 by addition o aqueouC; 5N NaOH. The solutlon was applied to a 2~8 x 59 cm. Michel-~iller glass colu~ packed wlt~ 25-40 microrl LiChroprep RP-8, and the column was eluted at 4 ml./min., using an FMI pump, with the same solvent combination used for sa~ple dissolution. The eluate was monitored at 254 nm usin~
an ISC~ Model UA-5 W detector. Selected 27-ml. fractions were analy2ed for the presence of factor D by analytical HPLC or. a 4.6 x 150 mmO stainless steel column packed in our laboratoris with 10 micrGn Nucleosil C18. The 1~ sample was applied using a Rheodyne Model 7120 in-jec~ion valve. The same solvent combination used for the prepar~tive elution was supplied at 0.6 ml.~min. by a Milton Roy Du~lex 1`~1inipum?. Factor D W25 detected at 225 ~m usin~ an ISCO Model lg00 variable wavelength W
detector. The portion of the eluate from 405-1134 ml.
was concentra~ed under reduced pxessure to a volume of 500 ml., and desalted on a column containing Diaion H~-20 resin in the fashion previously described~ The eluate was conc~ntrat~d under reduced pressure and 2~ lyophilized to give 120 ~g. of factor D.
. Example 7 Isolation of A41030 Factor E
A 0.3 g. portion o~ the A41030 complex was dissoi~ed in 30 ml. oS a solv~nt consistlng of ~ater:
ac~toni~rile:~odi~m chlorid~ (~5:1i:2 g.~L.), and applied to a 2 . 8 x ~3 cm. Mi chel-Miller glass column pac~ced in our lab~ratories with 25-4C F;icron LiChroprep ~-8.
~n FMI pwmp was u5ed to elute thQ colu~ at 12 ml~min~
3~ (85 psi~ with t:~e same solven~ combi~ation used for . . ... .. ., .. ; .. .. ,,, ,,,, , , ,,,,,,, , , ,,, ., ...... . ... . .. .. ~, ... . . .... .......... ........ .. . .. . . .. .... . .. . .... ..
...

X-5984 ~55~

sample dissolution, collectin~ ~4-ml. frac-tions. The eluate was monitored a~ 254 nm using an ISCO Model UA~5 W detector. Fractions .1-54, inclusi~, we~e dis-carded. Fractions 55-122, inclusive, rich in factsr E, were combined and concen~ra~ed under reduced pressure 5 to a ~olume of 50 ml.
Concentrates from 13 similar purifications were combined, diluted to l.5 L. with water, and applied at 5 ml./min. to lO0-ml. of Diaion HP-20 res7n in a column (2.8 x 22 cm.), pre~iously equili~rated with l~ water~ The column was washed with water (900 ml.3 until no chlorlde was de'ected in the wash by precip-itation as silver chloride. E~ution was then performed with water:methanol (l:l) at lO ml./minO~ collecting 300-ml. fractions. Fractions were anal~e~ ~or activity - ~ against 3. sub~lis. Fractions 1-8, inclusive, were.
combined, concentrated under reduced pressure, and lyophilized to give l.04 g. of a factor E-enriched mixture of factors. A 0.5 g. portion of this mi~tur~
was dissolved in lG ml~ of a solvent consisting of water:acetonitril~:sodium chloride ~84:l4:~ s.~L.), and applied to a 2.3 x 59 cm. Michel~ iller glass col~mr.
packed with ~5~40 micron LiChroprep RP-8. ~n FMI pu~p was used to elute the column at 5 ml./min., with the same solvent combiIIation used for sample dissolution, and 25~ml. frac~ions wer~ collected~ The elua~e was monitor~d at 254 nm using ~n TSCO Model UA-5 W
detector. Selec~ed fractions were analyzed ~or ~he presence o~ ractor E by analytical HP~C on a 4.6 x 150 mm~ st2lnless steel colu~ pacXed in our laborato~ies .~ith 5 micron ODS-Hyperspheres (Shandoll Southern Produc~s, X-598~ ~57 Ltd., Cheshire, England). The sample was applied using a Rheodyne ~odel 7120 injection valve. The solvent, consisting of water:acetonitrile:sodi~m acetate (81:19:2 g./L.) adjusted to p~ 6 with gla~ial acetic acid, was supplied at 0.65 ml./min. by a Milton Roy Duplex S Minipump. Factor E was detected at 225 nm usin~ an ISC0 Model lB00 variable wavelength W detector, The portion of the eluate from 1520-1780 ml. was concen-trated under reduced pressure to a volume of 50 ml.
Concentrates rom three similar purifications we_e combined, diluted to 1 L. with wa~er, and applied at 10 ml./min. to 100-ml. of Diaion HP-20 resin in a column (2.8 x 22 cm.), p~eviously equilibrated with water. The column was washed witn water (200 ml.) adjusted Wi! h aqueous formic acid to pH 2.5, unLil no ., chloride was detected in the wash by precipitation as silver chloride. Elu~ion was performed with 0.5 L. of water acetonitrile (6:4) at 15 ml ~ /min . The eluate was concentrated under reduced pressure and ~yophili~ed to gi~e 202.2 mg. of partially purlried factcr ~. This preparation was dissolved in 4 ml. of a solvent con-sisting o. w~t~r:acet~ni~rile:sodium chloride (8~:14:2 g./L.) and chromatographed at 4 ml./min~ on a 2.8 x 59 cm.
Michel-~iller ~lass colu~n, packed with 25-40 micron LiChroprep ~P-~ as previously describa~. The portion o~ the eluate from ~060-2~80 ml., rich in ~actor E, was conc~ntrated under reduced p~essure to a volume of 50 ml. Concentrates Sr~m th-~e similar ~urifications wexe combined and desalted cn 100-ml. of Diaion HP-~0 resin ~n a column, as p-eviously described. The eluat~
~as c~ncent~at~ under reduced pressure and lyophilized t~ ~ive 242 mg. o ~ctor E~

,, .. ~, .. ........... . ........... ... . ......

9~

X-59~4 ~58-Example 8 Isolation of A41030 Factor ~
A 9.0 g. portion of the A41030 complex was dissolved in 200 ml. of a solvent consisting of water~
ace~oni~rile:sodium chlorid~ (83:17:2 g./L.) a~d t~e solution wa~ filtered. The filtrate was applied to an 8 x 100 cm. stainless steel column p~cked with 4 L~ of 10-20 micron ~P-l/C18 reversed-phase silic~ gel which was prepared in ou- laboratorles ~y the special pro-10 c~dure descri~ed in Example 3. Tne column, par~ of aChrom2tospac Prep-100 unit, was eluted at 60 ml./mirl., with the same solvent combination used for sample dis301ution~ and 480~ml. fractions were colla~ted. The eluat~ was monltored at 254 nm using an ISCO Model UA-5 UV detec~or. Selected fractions were analyzed for the presQ~c~ of fac~o_ F by analytical HPLPLC on an 0.8 x 30 cm. Michel-~liller glass column packed in our laboratories with ~5 40 micron LiChrosrep RP-8. The solvent, water.acetonitrile:sodium chloride ~84:16-2 g-~L~), was supplied at 4 ml./min. by an ~lï pump.
Factor F was detected 2t 254 nm using an ISCO Model UA-5 UV detector. Fxactions 1-25, inclusive, were discarded. Fractions 26-36, inclusive, rich in factor F, were combined nd concentrat~d unde~ reduced pressure 2~ ~ a ~olume of about 500 ml.
Concen'rates from three similar purifications were combined, fil~ered, and ~he filtrate ~pplied at 10 ml./min~ to 100 ml. of Diaion HP~20 resin in a column ~2 . 8 x 22 cm. ), previously equilibrated with 30 water . The columrl was ~ashed with .4ater (900 ml . ~

......................... .... ..... ....................................................................................................................................... .....
........................

X-5g8~ ~59-until no chloride was detected in the wash by precip-itation as silver chloride. Elutlon was performed with l L. of water:acetonitrile (6:4) at 15 ml . /min . Th~
eluate was concentrated under reduced pr~ssure a.nd lyophilized to give ~.6 g. o partially puxifled factor F.
A 500 mg. por~ion of this preparation was dissolved in 10 ml. of a solvent consisting of water:acetonitrile:
sodium ~hlorlde (84:16:2 s.~L.), by adjustment to pH
7.0 with aqueous sodi~m hydroxide. The solution was ~pplied tc a 4.7 x 45 cm. Michel-Miller glass column ~ packed in our laboratories wi h 25-~0 micron LiChroprep RP~18. An Fl~lI pump was us~d to elute the column at ~ ml./min., with the s~le solvent combination used for sample dissolution, a~ 24-ml. fracticns were collected.
The eluate was.monitored at 254 nm using an ISCO Model UA-5 W detector. Selected fractions were analyzed for the presence of .actor ~, usins the analytical HPLPLC
s~stem previously descri~ed. The portion of the eluate from 1940-2520 ml., rich in factor Ft was concentrate~
undex reduced pressure to a volume or about 300 ml.
Concentrates from two similar puri~ications were combin~d and applied at iO ml./m.in. to lO0-ml. of Diaion HP-20 resin in a column (2.8 ~ ~2 cm.), previously equilibrated with water. The column was washed wi ~h water (300 ml.) adjusted to DH 2.5 with rormic acidr until no cnloride was detected in th~ wash by precip-itation as silv~x chloride. ~lution was perfo~med with 0.75 L. of waterOacetonirrile ~6O4). The eluate was concentrated under re~uced pressu e and lvophilized to si~e ~99 ~g. of factor ~.

..... .. ,.. .... . .... ......... ... .. ................................ ....... ............................... .. ... ... ~................ ....

~-5984 ~60-Example 9 Isolation of A41030 Factor G
An 8 g. portion of the A~1030 comple~ from Example 2 was dissolved in 2~0 ml. o a solven~ con-sisting of w~ter:acetonitxile:sodium chloride ~84:16:2g./L.~ and filtered. Th~ fi.ltrate was applied to a ~tainless steel colu~n (8 x 100 cm.) packed with 4 L.
of 10 20 micron LP-l/C18 r~versed phase silic~ gel which ~as prepared in our laboratories by the special 1~ proceduxe described in Example 30 The colu~ was part of a Chromatospac Prep-100 unit (see Example 3). The column was eluted at 60 ml./min. with water:aceto-r~itrile:sodium chloride (84:16:2 g./L.), collecting 4~0-ml. fxactions. The eluate was monitored a. 254 nm as de.~crib~d in Ex~ple 3. Selected f:-a~tions werP
anal~zed ~or the pre~ence Ot factor G by an analy~ical hi~h p rEormance liquid chromatography (HPLC) procedure descri~ed in preceding Examples.
Fractions Z2-35, inclusive, rich in ~actor G, 2~ were combined and concentrated under reduced pressu~e to a volume of 50Q ml. Concentrates from three similar purifications were com'~ined, adjusted to pH 8.5 with aqueous sodium hydroxide, and filtered. The filtrate was applied at lC ml./min. ~o 100 ml. of Diaion HP-~0 resin in a column ~2.8 ~ 22 cm~, pre-viously ~quilibrated with water. The column was washed with water ~400 ml. adjusted to pH 2.5 with 40r~ic acid) until no chlo~ide wa~ etected in the wash bv precipit~tior. 2S sil~er chloride. Elution was perfoxmed ~ith water:acetcr,itrlle (6:4) a~ 15 ml./min./

~-598~ -61-collectins 1 L. fractions. Fractions were a~lyxed for activi~y against B. su~tilis. The active fractions were combined, concentrated under reduced pressure, and lyophilized to give 2.85 g. of matexial.
A 0.5 g. portion of this material was dis-5 solved in 10 ml. of A solvent consisting of water:
acetonitril~:dibutylamine (80:20:0.03~, which so~vent had been 2djusted to p~ 7.8 with phosphoric acid) by addition of dibutylamine until solution had been accomplished (final pH ~.2)~ The solution was appl~ed to a 2.8 x 5~ cm. Michel Miiler HPLPLC glass column packed with 25-4Q micron LiChroprep RP-8 (from MC/B
ManuCacturing Chemlst, Inc., Cincinnati, OH).
Usi~s an FMl pump~ the column was eluted at ~ ml./m1n~wlth the same solver.. combinatio~ used ~or sample ~issolu~ion. The eluat~ was monitored at 254 ~m using An ISCO ~odel ~-5 W detector. Selected 10 ml~
fractions were analy~ed for the presence of act~r G
by ~he ar.alytical HPLC procedure described in pr~cedir,g Examples.
~ ~ractions 54-74, inclusive, rich i~ .actor ~, were comblned t~ith fractions from ~wo similar purifi-ca~ions and applied at 10 ml./min. to 100 ml. of Diaion HP-~O resin in a column (~.8 x 22 cm), pre-viously equilibrated with water. The column was washed wit~ waker (300 ~.1.) adjusted to pY. 2.5 with formic acid, until ns chloride was detecte~ ir. the wash by precipitation as silver chloride. Elution was perfo~led with 0.75 L. or water:acetoni~rile ~604).
The Plua~e was concentrated under reduced pr2~sure and iyophilir~ed to give 960 mg~ of '~actor G.

2~3q;~
A

Example 10 Sample Preparation for Biological Assay and Quantitative Analysis of A41030 Factor A in Dried Whole Broth One liter of whole broth was concentrated to a volume of 200 ml. and lyophilized to give 31.5 g. of dried whole broth. A 400 mg. sample of the dried wholë broth was extracted 3 times with 10 ml portions of water at pH
8.5. The extracts were combined, concentrated to a volume of 10 ml~, and portions of this concentrate used for bio-logical assay. The turbidimetric assay was conducted on a semiautomated system (Autoturb~ microbiological assay system, Elanco) described by N~R. Ku~el and F~o Kavanaugh in J. Pharmaceut. Sci. 60~, 764 and 767 (1971). In testing the A41030 complex, the following test parameters were used: Staphylococcus aureus ATCC 9144 in a n~trient broth medium (pH 7), incubated for four hours at 37C.
Test samples and standard were dissolved in methanol:water (l l)o The standard, A41030 factor A, was presented to the Autotur ~ carrousel at concentrations of 0.4, 0.S~
0O9y 1.2, and 1O5 mcg./ml.
One milliliter of the above concentrate was purified by the following procedure to be used for analysis by ~PLCo (a) One C-18 SEP-PA ~ cartridge (silica gel cartridge, Waters Associates, Inc., ~ilford, Mass~) was washed with 10 ml. of methanol, using a 10 ml. syringe with a Luer fitting, as known to the art.
(b) ~ash the same cartridge with 10 ml. of water.

X-59~4 -63-(c) Apply 1 ml. of the c:oncentrate from above to the cartridge at the rate of approximately 1 ml.~min.
(d) Wash the cartridge with 1 ml. of water and blow the cartridge dry.
~e) Elute the cartridge with 1 ml. of a solution of tetrahydrofuran:water (1~1) at about 0.5 ml./min.
(f) Remove the tetrahydrofuran fro~ the eluate ln 1~ vacuo, or alternatively, under a nitrogen ___ stream, and reconstitute the elua-te to a volume of 1 ml. with water.
(~) Analy~e the solution by HPLC procedure as - described hereinbefore.
lS The resul(~s of the assay for biological activity and the HPI.C analysis of the whole broth are recorded in Ta'Dle 14, which follows.

3~

T~ble 14 Biolo~ical ~ctivity and HPLC Anal ~sis o~
A41û30h in Who1 e Broth Conc2~ltr. Total Wte ~o~al Wt ~f + of of S~l~lple ~oO Wt. A41030A Activity* A41030A A41030 106.3 kg ~.42 mg,fg ~91 g 472 g 9~.1 146. 5 kg 10. 8 mg~g }~5 ~ 1582 g 93. 9 ~ 3tal l~iological activity cGmprise~ of A4103Q ~actors A, B, C, D, E, F and c~
G., As det:er~ ned by HPLC.

~ t,-

Claims (20)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing A41030 complex, or Factor A, B, C, D, E, F, or G, or a pharmaceutically-acceptable salt thereof, which comprises cultivating Streptomyces virginiae NRRL 12525, in a culture medium containing assimilable sources of carbon, nitroqen, and inorganic salts under submerged aerobic fermentation conditions.

2. A method according to claim 1 followed by separation of the complex from the culture medium.

3. A method according to claim 2, in which the factors A, B, C, D, E, F or G are isolated from the complex.

4. A process according to claim 3 for preparing A41030 factor A of structure:

or a pharmaceutically-acceptable salt thereof.

5. A process according to claim 3 for preparing A41030 factor B of structure:

or a pharmaceutically-acceptable salt thereof.

6. A process according to claim 3 for preparing A41030 factor C of structure:

or a pharmaceutically-acceptable salt thereof.

7. A process according to claim 3 for preparing antibiotic A41030 factor D, which is a white, amorphous solid, having:
(a) an approximate elemental analysis of 54.46%
carbon, 4.35% hydrogen, 7.58% nitrogen, 4.27%
chlorine, and by difference, 29.34% oxygen;
(b) an observed molecular weight of about 1326, as determined by fast atom bombardment mass spectrometry;

(c) an infrared absorption spectrum having the following distinguishable maxima: 3448-3226 (strong, broad), 2959 (weak), 1661 (strong), 1592. (strong), 1511 (strong), 1429 (weak), 1290 (weak), 1227 (weak), 1212 (medium), 1163 (weak), 1143 (weak), 1053 (medium), and 1010 (strong) cm-1;
(d) ultraviolet absorption spectra with an absorption maximum, in acidic or neutral methanol:water (1:1), at 278 nm (.epsilon. 10,600) and, in basic methanol:water (1:1), at 298 nm (.epsilon.
19,900);
(e) two titratable groups in 66% aqueous di-methylformamide with pKa values of about 5.5 and 7.6;
(f) is soluble in alcohol-water mixtures, in dimethylsulfoxide, in dimethylformamide, in dimethylsulfoxide-water mixtures, in di-methylformamide-water mixtures, in dilute aqueous acid, or in dilute aqueous base; and (g) the pharmaceutically-acceptable, non-toxic salts of said A41030 factor D.

8. A process accordlng to claim 3 for preparing A41030 factor E of structure:

or a pharmaceutically-acceptable salt thereof.

9. A process according to claim 3 for preparing A41030 factor F of structure:

or a pharmaceutically-acceptable salt thereof.

10, A process according to claim 3 for preparing antibiotic A41030 factor G, which is a white solid having:
(a) an approximate elemental analysis of 50.02%
carbon, 4.61% hydrogen, 4.74% chlorine, 6.11%
nitrogen, and 30.70% oxygen;
(b) an observed molecular weight of about 1684, as determined by fast atom bombardment mass spectrometry;
(c) an infrared absorption spectrum having the following distinguishable absorption maxima:

3320 (very broad, strong), 2975 (sharp, weak), 2920 (sharp, weak), 1659 (normal, strong), 1594 (broad, strong), 1512 (sharp, strong), 1492 (shoulder), 1430 (sharp, weak), 1386 (broad, weak), 1337 (broad, weak), 1308 (sharp, weak), 1264 (sharp, weak), 1230 (broad, medium), 1145 (broad, medium), 1077 (sharp, medium), 1062 (sharp, medium), 1014 (sharp , medium), and 846 (broad, medium) cm-1;
(d) ultraviolet absorption spectra with an absorption maximum, in acidic or neutral methanol:water (1:1), at 278 nm (.epsilon. 15,000) and, in basic methanol:water (1:1), at 298 nm (.epsilon.
18,000);
(e) two titratable groups in 66% aqueous di-methylformamide with pKa values of about 5.4 and 7.0;
(f) is soluble in alcohol-water mixtures, in dimethylsulfoxide, in dimethylformamide, in dimethylsulfoxide-water mixtures, in dimethylformamide-water mixtures, in dilute aqueous acid; or in dilute aqueous base; and (g) the pharmaceutically-acceptable, non toxic salts of said A41030 factor G.

11. A41030 antibiotic complex or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 2, or by an obvious chemical or biological equivalent thereof.

12. A41030 antibiotic factor A or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 4, or by an obvious chemical or biological equivalent thereof.

13. A41030 antibiotic factor B or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 5, or by an obvious chemical or biological equivalent thereof.

14. A41030 antibiotic factor C or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 6, or by an obvious chemical or biological equivalent thereof.

15. A41030 antibiotic factor D or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 7, or by an obvious chemical or biological equivalent thereof.

16. A41030 antibiotic factor E or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 8, or by an obvious chemical or biological equivalent thereof.

17. A41030 antibiotic factor F or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 9, or by an obvious chemical or biological equivalent thereof.

18. A41030 antibiotic factor G or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 10, or by an obvious chemical or biological equivalent thereof.

19. The axenic culture of the microorganism Streptomyces virginiae NRRL 12525.

20. Streptomyces virginiae NRRL 12525.